Increasing salt tolerance in plants by overexpression of vacuolar Na+/H+ transporters

a technology of vacuolar na +/h + and plant salt, which is applied in the direction of plant/algae/fungi/lichens, peptides, tissue culture, etc., can solve the problems of large areas of the indian subcontinent being rendered unproductive, destroying ancient and recent agrarian societies, and extreme cost of measures

Inactive Publication Date: 2005-07-14
BLUMWALD EDUARDO +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Environmental stress due to salinity is one of the most serious factors limiting the productivity of agricultural crops, which are predominantly sensitive to the presence of high concentrations of salts in the soil.
Salt impinging on agricultural soils has created instability and has frequently destroyed ancient and recent agrarian societies.
Large areas of the Indian subcontinent have been rendered unproductive through salt accumulation and poor irrigation practices.
Although there is engineering technology available to combat this problem, though drainage and supply of high quality water, these measures are extremely costly.
In most of the cases, due to the increased need for extensive agriculture, neither improved irrigation efficiency nor the installation of drainage systems is applicable.
These soils are inherently high in salt and require vast amounts of irrigation to become productive.
One of the main disadvantages of using this gene for transformation of plants is associated with the typical problems encountered in heterologous gene expression, i.e. incorrect folding of the gene product, targeting of the protein to the target membrane and regulation of the protein function.
However, enzymes extracted from these plants show high sensitivity to salt.

Method used

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  • Increasing salt tolerance in plants by overexpression of vacuolar Na+/H+ transporters
  • Increasing salt tolerance in plants by overexpression of vacuolar Na+/H+ transporters
  • Increasing salt tolerance in plants by overexpression of vacuolar Na+/H+ transporters

Examples

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example 1

Preparation of Polyclonal and Monoclonal Antibodies.

[0170] Hydropathy profiles of the Arabidopsis Na+ / H+ antiport revealed a relatively hydrophilic domain (at the C-terminus) with possible regulatory functions. The C-terminus was sub-cloned into the pGEX—2TK vector (Pharmacia) to allow the overexpression of the C-terminus polypeptide as a GST-fusion polypeptide in E. coli. The fusion polypeptide was purified by glutathione-affinity chromatography and used as an antigen in rabbits to obtain polyclonal antibodies (30).

[0171] Monoclonal antibodies are prepared in mice hybridomas according to established techniques (30) using the C-terminus polypeptide as described above. Polyclonal and monoclonal antibodies raised against other regulatory regions of the Arabidopsis Na+ / H+ antiport are also obtained as described above. The invention includes the antibodies and the hybridoma which secretes the monoclonal antibodies.

example 2

Identification of Homologous Nucleic Acid Molecules from Other Plant Species, Preferably Salt Tolerant Species.

[0172] Several experimental approaches are used to identify homologous nucleic acid molecules from salt tolerant species. a) We screen cDNA and genomic libraries from sugar beets (a moderate salt-tolerant crop, also known as red beet) under low-stringency conditions with an Arabidopsis Na+ / H+ antiport cDNA as a probe (31); b) We apply PCR techniques using degenerate oligonucleotide primers designed according to the conserved regions of the Arabidopsis Na+ / H+ antiport (32); c) We screen cDNA expression libraries from different plants (salt-tolerant and salt-sensitive) using antibodies raised against an Arabidopsis Na+ / H+ antiport (31). We also use bioinformatics techniques to identify nucleic acid molecules. The invention includes methods of using such a nucleic acid molecule, for example to express a recombinant polypeptide in a transformed cell.

[0173] The techniques des...

example 3

Overexpression of the PNHX Transporter Preferably Arabidopsis Transporter (AtNHX).

[0174] The Na+ / H+ antiport is expressed in Arabidopsis plants, although the wild type plants show impaired growth at NaCl concentrations higher than 75 mM. The Na+ / H+ antiport is overexpressed in these plants in order to improve their tolerance to high salt concentrations. A full length cDNA (preferably coding for the AtNHX1 polypeptide (AtNHX2, AtNHX3 or AtNHX4) cloned from an Arabidopsis thaliana (Columbia) seedling cDNA library is ligated into a pB1NS1 vector (33). This vector contains a constitutively strong promotor (“super-promotor” (20)). Also, T-DNA vectors (pBECKS) are used (34). Constructs containing the AtNHX1 cDNA with the full Na+ / H+ antiport open reading frame in a sense orientation were selected by colony hybridization using the AtNHX1 as a probe and by restriction-digest analysis using BglII restriction endonuclease. These constructs are used to transform Agrobacterium tumefaciens, an...

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Abstract

The invention is isolated nucleic acid molecules encoding Na+ / H+ exchanger polypeptides for extrusion of monovalent cations from the cytosol of cells to provide the cell with increased salt tolerance. Crop species transformed with the nucleic acid molecule are capable of surviving in soil with high salt levels that would normally inhibit growth of the crop species.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional application of U.S. patent application Ser. No. 10 / 155,535 filed May 24, 2002, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 271,584, filed Mar. 18, 1999, which claims priority to U.S. Patent Application Ser. Nos. 60 / 116,111, filed Jan. 15, 1999, and 60 / 078,474, filed Mar. 18, 1998, each of which is hereby incorporated in its entirety.BACKGROUND OF THE INVENTION [0002] Environmental stress due to salinity is one of the most serious factors limiting the productivity of agricultural crops, which are predominantly sensitive to the presence of high concentrations of salts in the soil. Large terrestrial areas of the world are affected by levels of salt inimical to plant growth. It is estimated that 35-45% of the 279 million hectares of land under irrigation is presently affected by salinity. This is exclusive of the regions classified as arid and desert lands, (which comprises 25% of ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/415C12N15/82
CPCC12N15/8273C07K14/415
Inventor BLUMWALD, EDUARDOAPSE, MARIS
Owner BLUMWALD EDUARDO
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